WO2022196714A1

WO2022196714A1 - Pericyte having basic fibroblast growth factor (bfgf) gene introduced therein

Info

Publication number: WO2022196714A1
Application number: PCT/JP2022/011788
Authority: WO
Inventors: 憲一郎嶋谷; 照佐藤; 雅夫笹井; 芳樹澤; 充弘齋藤; 繁宮川
Original assignee: アステラス製薬株式会社; 国立大学法人大阪大学
Priority date: 2021-03-17
Filing date: 2022-03-16
Publication date: 2022-09-22
Also published as: JPWO2022196714A1; EP4310176A1

Abstract

The present invention addresses the problem of providing a cell therapy expected to be useful as an angiogenic therapy for peripheral vessel diseases such as critical limb ischemia. Specifically, a pericyte having high angiogenic potentional and a method for producing the same are provided. A pericyte having a bFGF gene introduced therein and a method for producing the same are provided.

Description

Pericytes transfected with basic fibroblast growth factor (bFGF) gene

The present invention is characterized by pericytes into which a basic fibroblast growth factor (bFGF) gene has been introduced, pharmaceutical compositions containing the pericytes, methods for producing the pericytes, and administration of the pericytes. It relates to angiogenesis therapy and the like.

Capillaries connect arterioles and venules, and are distributed deep in the body tissue in a mesh pattern so that oxygen and nutrients can be supplied to every corner of the body's periphery. Capillaries are composed of a single layer of vascular endothelial cells and pericytes (vascular pericytes) surrounding them that form the luminal structure. Pericytes, as cells that coat vascular endothelial cells, play an important role in normal blood flow regulation such as maturation and stabilization of blood vessels and maintenance of the blood-brain barrier. Critical lower extremity ischemia is a serious disease that impairs blood flow, but no effective drug therapy has been established, and it is treated by bypass surgery, endovascular treatment, and the like. In recent years, there has been a demand for the development of a new therapeutic method that induces the formation of peripheral capillaries by cell therapy (Patent Document 1).

"Basic fibroblast growth factor (bFGF)" is a member of the growth factor family, also called FGF-2. It is known to contribute to angiogenesis by promoting endothelial cell growth and tube formation. Human bFGF/FGF-2 protein is a single-chain polypeptide with a molecular weight of 18 kD consisting of 154 amino acids, and is known to be released from macrophages, endothelial cells, damaged muscle fibers, etc. (Henke C et al., Am. J. Pathol., (1993), 143: 1189-1199, Wang YX et al., J. Cell Sci., (2014), 127: 4543-4548). The most characteristic action of bFGF/FGF-2 in angiogenesis is to directly act on vascular endothelial cells to promote proliferation and tube formation of vascular endothelial cells. It is also believed that bFGF/FGF-2 indirectly promotes angiogenesis by regulating the expression of vascular endothelial growth factor (VEGF) in vascular smooth muscle cells (Non-Patent Document 1). It has been reported that introduction of the bFGF/FGF-2 gene into ischemic muscle tissue using a Sendai virus vector enhances endogenous VEGF and hepatocyte growth factor (HGF) expression, and induces improvement in lower extremity ischemia. (Non-Patent Document 2). Thus, bFGF/FGF-2 is a potent angiogenic factor, and is expected to be applied to ischemic diseases.

WO2008/104064

The object of the present invention is to provide cell therapy that is expected to be useful as angiogenesis therapy for peripheral vascular diseases such as severe lower extremity ischemia. Specifically, the object is to provide pericytes with high angiogenic potential and a method for producing the same.

In order to solve the above problems, the present inventors conducted intensive studies such as introducing several genes encoding angiogenic factors into pericytes. , the angiogenic ability of the pericytes transplanted into the living body is remarkably enhanced, and furthermore, the pericytes are applicable to angiogenic therapy such as severe lower extremity ischemia. The present invention has been completed based on such findings.

Specifically, the present invention has the following features:
[1] Pericytes into which a basic fibroblast growth factor (bFGF) gene has been introduced.
[2] The pericyte according to [1], wherein the pericyte is primary pericyte.
[3] The pericytes according to [1], wherein the pericytes are pericyte-like cells differentiated from pluripotent stem cells.
[4] The pericyte of [3], wherein the pluripotent stem cells are human pluripotent stem cells.
[5] The pericyte of [3] or [4], wherein the pluripotent stem cells are embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells).
[6] A pharmaceutical composition for angiogenesis therapy, comprising the pericyte of any one of [1] to [5].
[7] The pharmaceutical composition of [6], wherein the angiogenesis therapy is treatment of critical limb ischemia.
[8] The pharmaceutical composition of [6] or [7], which is used in combination with vascular endothelial cells.
[9] A pharmaceutical composition for angiogenesis therapy, comprising a combination of the pericytes and vascular endothelial cells of any one of [1] to [5].
[10] The pharmaceutical composition of [9], wherein the angiogenesis therapy is treatment of critical limb ischemia.
[11] The method for producing pericyte according to any one of [1] to [5].
[12] An angiogenesis therapy comprising administering a therapeutically effective amount of the pericyte according to any one of [1] to [5] to a subject.
[13] The angiogenesis therapy of [12], further comprising administering vascular endothelial cells.
[14] The angiogenesis therapy of [12] or [13], which is a treatment for critical limb ischemia.
[15] Use of the pericyte according to any one of [1] to [5] in the manufacture of a pharmaceutical composition for angiogenesis therapy.
[16] Use of the pericyte according to any one of [1] to [5] in the manufacture of a pharmaceutical composition for treating critical limb ischemia.
[17] The pericyte of any one of [1] to [5] for use in angiogenesis therapy.
[18] The pericyte according to any one of [1] to [5], for use in treating critical limb ischemia.
[19] Pericytes with enhanced expression of endogenous bFGF.

In the present invention, by introducing the bFGF gene into pericytes, pericytes with high angiogenic potential can be obtained and produced. In addition, bFGF gene-introduced pericytes obtained by the method of the present invention can be used for angiogenesis therapy for severe lower extremity ischemia and the like.

FIG. 1 shows the results of evaluating the bFGF expression level in the bFGF gene-introduced human primary pericyte (bFGF-Primary pericyte) obtained in Example 3 together with the bFGF expression level in the human primary pericyte (Primary pericyte) in Example 4. is. The vertical axis indicates the bFGF expression level (ng/mL). Error bars indicate ± standard error of the mean. 2 shows the results of qualitative evaluation of the angiogenic potential of the bFGF gene-introduced human primary pericytes obtained in Example 3 in Example 5. FIG. Specifically, human umbilical vein endothelial cells (HUVEC), HUVEC and human primary pericyte (HUVEC), or HUVEC and bFGF transgenic human primary pericyte (bFGF-Primary pericyte/HUVEC) was administered to NOG mice together with Matrigel, Matrigel was recovered from the mice 14 days later, and photographed. 3 shows the results of quantitative evaluation of the angiogenic potential of the bFGF gene-introduced human primary pericytes obtained in Example 3 in Example 6. FIG. Specifically, each Matrigel collected by the procedure of Example 5 was crushed, and the concentration of hemoglobin contained in the supernatant after centrifugation was measured. The vertical axis indicates the hemoglobin concentration (μg/mL) contained in the Matrigel-derived supernatant. The P value indicated by ** in FIG. 3 is 0.0001. Error bars indicate ± standard error of the mean. FIG. 4 shows the therapeutic effect of administering the bFGF gene-introduced human primary pericytes obtained in Example 3 to lower limb ischemia model mice in Example 7. FIG. The horizontal axis in the left diagram of FIG. 4 indicates the number of weeks after administration of the control medium (Medium) or bFGF gene-introduced primary pericyte (bFGF-Primary pericyte) to the ischemic limb, and the vertical axis indicates blood flow in the ischemic limb. The blood flow ratio (%, Ischemic/normal) obtained by dividing the signal value of (Blood perfusion) by the signal value of the normal limb blood flow is shown. The vertical axis in the right diagram of FIG. 4 indicates the AUC (Area Under Curve) after administration of the control medium or primary pericytes into which the bFGF gene was introduced, calculated based on the left diagram. The P value indicated by ** in the right diagram of FIG. 4 is 0.0139. Error bars indicate ± standard error of the mean.

The present invention will be described in detail below.

<Pericytes into which the bFGF gene was introduced>
The present invention provides pericytes into which a bFGF gene has been introduced (also referred to as "pericytes of the present invention").

The pericytes of the present invention are pericytes into which the bFGF gene has been introduced. The nucleotide sequence of the bFGF gene and the amino acid sequence of bFGF are already known, and the sequences are published in public databases and the like. For example, the nucleotide sequence and amino acid sequence of human bFGF are published as GenBank Accession Number: M27968.1 and AAA52448.1, respectively. Specifically, human bFGF is encoded by the gene with GenBank Accession Number: M27968.1 (SEQ ID NO: 1) and has the amino acid sequence (SEQ ID NO: 2) indicated by GenBank Accession Number: AAA52448.1. In the present invention, the bFGF gene to be introduced into pericytes includes genes encoding naturally occurring bFGF and genes encoding functional variants of bFGF. In one embodiment, the bFGF gene introduced into pericytes in the present invention is at least 80% or more, preferably 85% or more, 90% of the amino acid sequence published as GenBank Accession Number: AAA52448.1 As described above, it is a gene encoding a protein having 95% or more or 98% or more identity and having a function as bFGF. In one embodiment, the bFGF gene to be introduced into pericytes in the present invention has 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acids deleted, substituted, A gene consisting of an inserted and/or added amino acid sequence and encoding a protein having the function of bFGF. In one embodiment, the bFGF gene introduced into pericytes in the present invention is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:2. Having a function as bFGF can be confirmed by a known method (Beenken A & Mohammadi M, Nat. Rev. Drug Discov., (2009), 8: 235-253). In one embodiment, the bFGF gene to be introduced into pericytes in the present invention may be a gene encoding bFGF or a variant thereof to which a secretory signal has been added. As the secretion signal, a secretion signal known to those skilled in the art can be used, and in one embodiment, the Bmp2/4 secretion signal (US Pat. No. 7,816,140) can be used. In one embodiment, the bFGF gene introduced into pericytes in the present invention is at least 80% or more, preferably 85% or more, 90% or more, 95% or more, or It is a gene encoding a protein having an identity of 98% or more and having a function as bFGF. In one embodiment, the bFGF gene introduced into pericytes in the present invention is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:4. In addition, in this specification, "bFGF" may be described as "FGF-2."

With respect to nucleic acid sequences or amino acid sequences, the term “identity” as used herein refers to the NEEDLE program (Needleman SB et al., J. Mol. Biol., (1970), 48: 443-453) provided by default by searching. means the value Identity obtained using the parameters Said parameters are as follows.
Gap penalty = 10
Extended penalty = 0.5
Matrix = EBLOSUM62

≪Pericyte≫
"Pericytes" are cells that surround microvessel walls or capillary walls in the brain, periphery, retina, or the like, and are also called vascular pericytes. Its function is as described above, and as a cell that coats vascular endothelial cells, it plays an important role in normal blood flow regulation such as maturation and stabilization of blood vessels and maintenance of the blood-brain barrier (Daneman R et al., Nature, (2010), 468: 562-568; Armulik A et al., Dev. Cell, (2011), 21: 193-215). When the cell function of pericytes is impaired, the original functions of pericytes (for example, stabilization of blood vessels, maintenance of blood flow, maintenance of the blood-brain barrier, maintenance of the blood-nerve barrier, etc.) are impaired, resulting in diabetic retinopathy, etc. leading to serious blood flow disorder-related diseases. In addition, the existence of cells called mesoangioblasts, which have the ability to differentiate into muscle and bone, has been clarified in pericytes derived from skeletal muscle (Gerli MFM et al., J. Vis. Exp. ., (2014), 83: 50523, Gerli MFM et al., Stem Cell Rep., (2019), 12: 461-473, Shimatani K et al., Am. J. Physiol. Heart Circ. Physiol., ( 2021), on line: https://doi.org/10.1152/ajpheart.00470.2020).

- First generation pericyte -
In one embodiment, the pericytes of the present invention are primary pericytes into which the bFGF gene has been introduced.

As used herein, the term "primary pericytes" means pericytes directly collected from an individual organism, or primary cultured cells or subcultured cells obtained by culturing and proliferating the pericytes in vitro. Methods for isolating and culturing primary pericytes from individual organisms are described, for example, in Quattrocelli M et al., Methods Mol. Biol., (2012), 798: 65-76. Primary pericytes in the present invention are not particularly limited, but in one embodiment, they are human primary pericytes. Human primary pericytes include, for example, human patients themselves, or primary pericytes having the same or substantially the same human leukocyte antigen (HLA) genotype as the transplant recipient from the viewpoint of preventing rejection. It is preferable to use the site. Here, the term "substantially identical" means that the HLA genotypes match the transplanted pericytes to the extent that an immunosuppressive agent can suppress an immune reaction. It is a pericyte having an HLA type in which 3 loci of B and HLA-DR or 4 loci including HLA-C are matched.

-Pericyte-like cells-
In one embodiment, the pericytes of the present invention are pericyte-like cells induced to differentiate from pluripotent stem cells into which the bFGF gene has been introduced.

As used herein, the term "pericyte-like cells" means cells that have been induced to differentiate from pluripotent stem cells and have properties similar to primary pericytes. It can be confirmed by a known method that pericyte-like cells have properties similar to those of pericytes (Armulik A et al., Dev. Cell, (2011), 21: 193-215). Differentiation induction from pluripotent stem cells to pericyte-like cells can be performed using methods known to those skilled in the art /0316094). For example, the induction of differentiation from pluripotent stem cells to pericyte-like cells can be performed using the method described in the section <<Method for Inducing Differentiation of Pluripotent Stem Cells into Pericyt-like Cells>> below.

≪Pluripotent stem cells≫
As used herein, the term "pluripotent stem cell" means a stem cell that has pluripotency capable of differentiating into cells with many different properties and morphologies that exist in a living body, and that also has proliferative potential. Preferred pluripotent stem cells in the present invention are human pluripotent stem cells. In one embodiment, the pericytes of the present invention are pericyte-like cells induced to differentiate from human pluripotent stem cells into which the bFGF gene has been introduced.

The pluripotent stem cells used in the present invention are not particularly limited. , germline stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), cultured fibroblasts and bone marrow stem cell-derived pluripotent cells (Multi -lineage differentiating stress enduring cells; Muse cells), etc. Preferred pluripotent stem cells for inducing the differentiation of pericyte-like cells in the present invention are ES cells or iPS cells. In one embodiment, the pericytes of the present invention are pericyte-like cells induced to differentiate from embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) into which a bFGF gene has been introduced. In one embodiment, the pericytes of the present invention are pericyte-like cells induced to differentiate from human ES cells or human iPS cells into which a bFGF gene has been introduced.

－ES cells－
ES cells are stem cells that are established from the inner cell mass of early embryos (for example, blastocysts) of mammals such as humans and mice and that have pluripotency and the ability to proliferate through self-renewal. ES cells can be established by removing the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Furthermore, cells can be maintained by subculturing using a medium supplemented with substances such as leukemia inhibitory factor (LIF) and bFGF. Human ES cells are prepared by known methods (for example, Suemori H et al., Biochem. Biophys. Res. Commun., (2006), 345: 926-932, Kawasaki H et al., Proc. Natl. Acad. Sci. USA , (2002), 99: 1580-1585).

－iPS cells－
iPS cells are a general term for pluripotent stem cell lines that are artificially induced by introducing specific genes into somatic cells that have lost their pluripotency. Methods for producing iPS cells are known in the art, and can be produced by introducing reprogramming factors into arbitrary somatic cells. Here, the initialization factors are, for example, Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15 Gene products such as -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1 are exemplified, and these reprogramming factors may be used alone or in combination. .初期化因子の組み合わせとしては、WO2007/069666； WO2008/118820； WO2009/007852；WO2009/032194； WO2009/058413； WO2009/057831； WO2009/075119； WO2009/079007；WO2009/091659； WO2009/101084； WO2009/ 101407； WO2009/102983； WO2009/114949； WO2009/117439； WO2009/126250； WO2009/126251； WO2009/126655； WO2009/157593； WO2010/009015； WO2010/033906； WO2010/033920； WO2010/042800； WO2010/050626； WO 2010/056831； WO2010/068955； WO2010/098419； WO2010/102267； WO2010/111409； WO2010/111422； WO2010/115050； WO2010/124290； WO2010/147395； WO2010/147612； Huangfu D et al., Nat. Biotechnol ., (2008), 26: 795-797; Shi Y et al., Cell Stem Cell, (2008), 2: 525-528; Eminli S et al., Stem Cells, (2008), 26: 2467-2474 Huangfu D et al., Nat. Biotechnol., (2008), 26: 1269-1275; Shi Y et al., Cell Stem Cell, (2008), 3: 568-574; Zhao Y et al., Cell Stem Cell, (2008), 3: 475-479; Marson A Cell Stem Cell, (2008), 3: 132-135; Feng B et al., Nat. Cell Biol., (2009), 11: 197-203; Judson RL et al., Nat. Biotechnol., (2009), 27: 459-461; Lyssiotis CA et al. USA, (2009), 106: 8912-8917; Kim JB et al., Nature, (2009), 461: 649-643; Ichida JK et al., Cell Stem Cell. , (2009), 5: 491-503; Heng JC et al., Cell Stem Cell, (2010), 6: 167-174; Han J et al., Nature, (2010), 463: 1096-1100; P et al., Stem Cells, (2010), 28: 713-720; Maekawa M et al., Nature, (2011), 474: 225-229.

The somatic cells used for the production of iPS cells may be either neonatal (offspring) somatic cells, healthy human or patient somatic cells, and are not particularly limited to these. In addition, any of primary cultured cells, passaged cells and established cells derived therefrom may be used. In one embodiment, the somatic cells used for the production of iPS cells are, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, (2) tissue progenitor cells, (3) ) Blood cells (peripheral blood cells, umbilical cord blood cells, etc.), muscle cells, skin cells, hair cells, liver cells, gastric mucosa cells, enterocytes, splenocytes, pancreatic cells, brain cells, lung cells, kidney cells, adipocytes, etc. They include differentiated cells present in organs and tissues.

When iPS cells are used as a material for inducing the differentiation of pericyte-like cells, the human leukocyte antigen (HLA) genotype of the transplant recipient is the same or substantially the same from the viewpoint of avoiding rejection. Cell-derived iPS cells are preferably used, but are not limited to this. Here, the term "substantially identical" means that the HLA genotypes match the transplanted cells to the extent that an immunosuppressive agent can suppress an immune reaction. and HLA-DR 3 loci or HLA-C plus 4 loci are matched HLA type iPS cells derived from somatic cells.

Allotransplantation of pluripotent stem cells, which are materials for inducing pericyte-like cells, prepared by the method described in, for example, Gornalusse GG et al., Nat. Biotechnol., (2017), 35: 765-772 It is also possible to use pluripotent stem cells that do not cause rejection in . The pluripotent stem cells that do not cause rejection in allotransplantation are preferably ES cells or iPS cells that do not cause rejection in allotransplantation, and human ES cells or human iPS cells that do not cause rejection in allotransplantation. is more preferred. In one embodiment, the pericytes of the present invention are pericyte-like cells induced to differentiate from pluripotent stem cells into which the bFGF gene has been introduced and which do not cause rejection in allotransplantation. In one embodiment, the pericytes of the present invention are pericyte-like cells induced to differentiate from human ES cells or human iPS cells into which the bFGF gene has been introduced and which do not cause rejection in allotransplantation.

<Pericytes with enhanced expression of endogenous bFGF>
In one embodiment, the pericytes of the present invention are pericytes in which endogenous bFGF expression is enhanced. Here, "the expression of endogenous bFGF is enhanced" means that in pericyte-like cells differentiated from primary pericytes or pluripotent stem cells, direct Both endogenous bFGF activity enhancement and indirect endogenous bFGF activity enhancement due to release of related inhibitory system, etc. are included. Enhancement of endogenous bFGF expression can be brought about by induction of endogenous bFGF expression by an exogenous factor. Methods for inducing endogenous bFGF expression include, for example, induction by acidosis (D'Arcangelo D et al., Circ. Res., (2000), 86: 312-318). The bFGF protein expression level in pericytes in which endogenous bFGF expression is enhanced is not particularly limited. In normal primary pericytes, bFGF expression was below the detection limit (see Example 4).

<Method for Producing Pericytes Introduced with the bFGF Gene>
The present invention also provides a method for producing bFGF gene-introduced pericytes (also referred to as “the production method of the present invention”).

In one embodiment, the production method of the present invention includes introducing the bFGF gene into pericytes. In one embodiment, the production method of the present invention includes introducing a bFGF gene into primary pericytes. In one embodiment, the production method of the present invention includes introducing a bFGF gene into pericyte-like cells that have been induced to differentiate from pluripotent stem cells. In one embodiment, the production method of the present invention includes introducing a bFGF gene into pericyte-like cells that have been induced to differentiate from ES cells or iPS cells. In one embodiment, the production method of the present invention includes introducing a bFGF gene into pericyte-like cells that have been induced to differentiate from human pluripotent stem cells. In one embodiment, the production method of the present invention includes introducing a bFGF gene into pericyte-like cells that have been induced to differentiate from human ES cells or human iPS cells.

In one embodiment, the production method of the present invention includes introducing a bFGF gene into pluripotent stem cells, and inducing differentiation of the bFGF gene-introduced pluripotent stem cells into pericyte-like cells. In one embodiment, the production method of the present invention comprises introducing a bFGF gene into ES cells or iPS cells, and inducing differentiation of the ES cells or iPS cells into which the bFGF gene has been introduced into pericyte-like cells. . In one embodiment, the production method of the present invention comprises introducing a bFGF gene into human pluripotent stem cells, and inducing differentiation of the human pluripotent stem cells into which the bFGF gene has been introduced into pericyte-like cells. . In one embodiment, the production method of the present invention comprises introducing a bFGF gene into human ES cells or human iPS cells, and inducing differentiation of the human ES cells or human iPS cells introduced with the bFGF gene into pericyte-like cells. including doing

[Method for introducing bFGF gene into cells]
The bFGF gene can be constructed using methods known in the art based on base sequence information. For example, the bFGF gene can be synthesized using gene synthesis methods known in the art.

In the present invention, the method of introducing the bFGF gene into pericytes or pluripotent stem cells includes methods commonly used for transfection of animal cells, such as calcium phosphate method, lipofection method, electroporation method, microinjection method, and viral vector. A method of introduction and the like can be used. In one embodiment, a method of introducing the bFGF gene into pericytes or pluripotent stem cells using a viral vector can be used. Viral vectors that can be used to introduce the bFGF gene into pericytes or pluripotent stem cells include lentivirus, adenovirus, adeno-associated virus or retrovirus. In one embodiment, a method of introducing the bFGF gene into pericytes or pluripotent stem cells using a lentiviral vector can be used. Specifically, as described in Example 3, the bFGF gene can be introduced into the cells by infecting the cells with a lentivirus for introducing the bFGF gene.

[Method for inducing differentiation of pluripotent stem cells into pericyte-like cells]
In the present invention, pericyte-like cells can be obtained by inducing differentiation from pluripotent stem cells into pericyt-like cells, as described above. The method for inducing the differentiation of pluripotent stem cells into pericyte-like cells is not particularly limited, but in one embodiment, it can be obtained by a method comprising the following steps (a) and (b):
(a) differentiating the pluripotent stem cells into early mesodermal cells; and
(b) differentiating the early mesodermal cells obtained in step (a) into pericyte-like cells;

Steps (a) and (b) are described below.
Step (a) is a step of differentiating pluripotent stem cells into primitive posterior mesoderms. In the present invention, the method of differentiating pluripotent stem cells into early mesodermal cells is not particularly limited, but for example, US9,868,939 and US9,771,561, Uenishi G et al., Stem Cell Reports, (2014), 3: 1073. -1084 can be used to induce differentiation from pluripotent stem cells to early mesoderm cells. The differentiation of pluripotent stem cells into early mesoderm cells has been described, for example, in Vodyanik MA et al., Cell Stem Cell, (2010), 7: 718-729, US9,771,561, and Uenishi G et al., Stem Cell Reports, (2014), 3: 1073-1084, can be confirmed using surface antigen markers specific to early mesodermal cells (PDGFRα, APLNR, etc.).

Step (b) is a step of inducing differentiation of the early mesodermal cells obtained in step (a) into pericyte-like cells. In the present invention, the method for differentiating early mesoderm cells into pericyte-like cells is not particularly limited, but, for example, the method of inducing differentiation from early mesoderm cells into pericyte-like cells described in US Pat. No. 9,868,939 is used. be able to. The differentiation of early mesodermal cells into pericyte-like cells can be confirmed using surface antigen markers such as NG2 and CD146 (Herrmann M. et al., Eur. Cells Mater., (2016) et al., Exp. Hematol., (2008), 36: 642-654, Lv FJ. et al., Stem Cells, (2014), 32: 1408-1419).

In one embodiment, in step (b), spheroids of early mesoderm cells are first formed, and then the spheroids of early mesoderm cells can be induced to differentiate into pericyte-like cells.

In the present invention, the method for forming spheroids of early mesodermal cells is not particularly limited, and known methods can be used. For example, a method using a methylcellulose medium described in US Pat. No. 9,771,561 can be mentioned. The composition of the spheroid-forming medium can be set with reference to known techniques (Vodyanik MA et al., Cell Stem Cell, (2010), 7: 718-729, etc.).

In the present invention, the method for differentiating spheroid-formed early mesoderm cells into pericyte-like cells is not particularly limited, and known methods can be used. For example, the method described in US9,868,939 can be used.

[Method for culturing bFGF gene-introduced pericytes]
The method for culturing/proliferating the pericytes of the present invention is not particularly limited, and a method for culturing/proliferating pericytes known in the art can be used.

The medium for culturing the pericytes of the present invention is not particularly limited as long as it is a medium suitable for culturing pericytes. ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI medium, StemSpan medium, StemPro medium and mixtures thereof can be used.

The medium for culturing the pericytes of the present invention may be appropriately supplemented with various nutrient sources necessary for cell maintenance and proliferation. For example, nutrient sources include carbon sources such as glycerol, glucose, fructose, sucrose, lactose, honey, starch, and dextrin; hydrocarbons such as fatty acids, oils, lecithin, and alcohols; ammonium sulfate, ammonium nitrate, ammonium chloride, urea , Nitrogen sources such as sodium nitrate, salt, potassium salts, phosphates, magnesium salts, calcium salts, iron salts, inorganic salts such as manganese salts, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, sulfuric acid It can contain ferrous iron, sodium molybdate, sodium tungstate and manganese sulfate, various vitamins, amino acids, and the like. In one embodiment, an amino acid such as glutamine can be used as a nutrient source added to the medium for culturing the pericytes of the present invention.

The medium for culturing the pericytes of the present invention may be appropriately supplemented with growth factors such as FGF family growth factors necessary for cell proliferation. The growth factor to be added is not particularly limited, but in one embodiment, bFGF or modified bFGF with enhanced thermostability can be used as the growth factor to be added to the medium for culturing the pericytes of the present invention.

The pH of the medium for culturing the pericytes of the present invention is in the range of 5.5-9.0, preferably 6.0-8.0, more preferably 6.5-7.5.

Pericytes are adherent cells that have the property of adhering to the extracellular matrix and proliferating. Therefore, in one embodiment, a suitable scaffold can be used in culturing the pericytes of the present invention. The scaffold is not particularly limited as long as it is a matrix, substrate, or carrier to which cells can attach and divide/proliferate. Examples include poly-L-lysine, poly-D-lysine, and the like. In one embodiment, a collagen matrix can be used as a scaffold for use in culturing pericytes of the present invention.

Cultivation of pericytes of the present invention can be carried out at 36°C to 38°C in one embodiment. In one embodiment, the pericytes of the present invention can be cultured at 36.5°C to 37.5°C in an atmosphere of 1% to 25% O ₂ and 1% to 15% CO ₂ while exchanging the medium as appropriate. can.

<Pharmaceutical composition, etc. of the present invention>
The present invention also provides pharmaceutical compositions comprising the pericytes of the present invention (also referred to as "pharmaceutical compositions of the present invention"). The pharmaceutical composition can be prepared by a commonly used method using excipients commonly used in the field, ie, pharmaceutical excipients, pharmaceutical carriers, and the like. In formulating the pharmaceutical composition, excipients, carriers, additives and the like according to these dosage forms can be used within a pharmaceutically acceptable range. In one embodiment, the pharmaceutical composition of the present invention comprises primary pericytes into which the bFGF gene has been introduced. In one embodiment, the pharmaceutical composition of the present invention contains pericyte-like cells induced to differentiate from pluripotent stem cells into which the bFGF gene has been introduced. The pharmaceutical composition of the present invention contains pericyte-like cells differentiated from ES cells or iPS cells into which a bFGF gene has been introduced. In one embodiment, the pharmaceutical composition of the present invention contains pericyte-like cells induced to differentiate from human pluripotent stem cells into which a bFGF gene has been introduced. In one embodiment, the pharmaceutical composition of the present invention contains pericyte-like cells induced to differentiate from human ES cells or human iPS cells into which a bFGF gene has been introduced.

In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for angiogenesis therapy. The pharmaceutical composition includes a therapeutic agent for angiogenesis therapy containing pericytes of the present invention. Angiogenesis therapy refers to the development of new cells in an ischemic organ, tissue, or body part to increase the amount of oxygen-rich blood reaching the ischemic organ, tissue, or body part. It is a therapeutic method that promotes the formation of blood vessels. Angiogenic therapies include treatment of peripheral vascular diseases such as critical limb ischemia and diabetic retinopathy, and diseases such as pulmonary hypertension. In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for treatment of critical limb ischemia, peripheral vascular diseases such as diabetic retinopathy, pulmonary hypertension, and the like. In one embodiment, the pharmaceutical composition of the invention is a pharmaceutical composition for the treatment of critical limb ischemia.

The present invention provides use of the pericyte of the present invention in the manufacture of a pharmaceutical composition for angiogenesis therapy. The invention provides pericytes of the invention for use in angiogenesis therapy. The invention also provides the use of the pericytes of the invention for angiogenesis therapy.

The present invention provides use of the pericyte of the present invention in the manufacture of a pharmaceutical composition for treating critical limb ischemia. The present invention provides pericytes of the present invention for use in treating critical limb ischemia. The present invention also provides use of the pericytes of the present invention for the treatment of critical limb ischemia.

The present invention also provides an angiogenesis therapy (also referred to as "the treatment method of the present invention") comprising administering a therapeutically effective amount of the pericyte of the present invention to a subject. In the treatment methods of the present invention, a "subject" is a human or other animal in need of such treatment. In one embodiment, a "subject" is a human in need of the method of treatment. When the pericyte of the present invention is administered to humans, it can be administered to the subject in the form of a pharmaceutical composition containing the pericyte of the present invention and a pharmaceutically acceptable excipient. The dosage and frequency of administration of the pharmaceutical composition of the present invention to humans can be appropriately adjusted according to the disease to be treated, its severity, and the age, weight and condition of the person to be treated.

The administration method of the pharmaceutical composition of the present invention is not particularly limited, and depending on the site of application, local transplantation by surgical means, intravenous administration, leg puncture administration, local injection administration, subcutaneous administration, intradermal administration, intramuscular administration. etc. can be considered.

The pharmaceutical composition of the present invention may be made into a sheet and applied directly to the affected area. The sheet may contain a suitable support as well as cells.

The pharmaceutical composition of the present invention may contain scaffolding materials and components that assist in cell maintenance/proliferation, administration to affected areas, and other pharmaceutically acceptable carriers. Components necessary for maintenance and growth of cells include medium components such as carbon sources, nitrogen sources, vitamins, minerals, salts, various cytokines, and extracellular matrix preparations such as matrigel.

The pericyte of the present invention or the pharmaceutical composition of the present invention can also be used in combination with vascular endothelial cells. As used herein, the term "combination" means administration of multiple active pharmaceutical ingredients simultaneously or separately to the same subject. In combination, the multiple active pharmaceutical ingredients may be contained in the same composition, or may be contained separately in different compositions. In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition used in combination with vascular endothelial cells. In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition further comprising vascular endothelial cells. In one embodiment, the therapeutic method of the present invention further comprises administering vascular endothelial cells.

The present invention also includes a pharmaceutical composition comprising a combination of the pericytes of the present invention and vascular endothelial cells. As used herein, the term "combination" means that multiple types of active pharmaceutical ingredients are contained in the same pharmaceutical composition, or multiple types of active pharmaceutical ingredients are contained separately in different pharmaceutical compositions. means that In one embodiment, the pharmaceutical composition obtained by combining the pericytes of the present invention and vascular endothelial cells is a pharmaceutical composition comprising the pericytes of the present invention and vascular endothelial cells. In one embodiment, a pharmaceutical composition comprising a combination of the pericytes of the present invention and vascular endothelial cells is a pharmaceutical composition in which the pericytes of the present invention and vascular endothelial cells are separately contained in different pharmaceutical compositions. It's a combination. In one embodiment, the pharmaceutical composition obtained by combining the pericytes of the present invention and vascular endothelial cells is a combination of the pharmaceutical composition containing the pericytes of the present invention and the pharmaceutical composition containing vascular endothelial cells. In one embodiment, the pharmaceutical composition comprising a combination of pericytes and vascular endothelial cells of the present invention is a pharmaceutical composition for angiogenesis therapy. In one embodiment, the pharmaceutical composition comprising a combination of pericytes and vascular endothelial cells of the present invention is a pharmaceutical composition for treating critical limb ischemia.

≪Vascular endothelial cell≫
“Vascular endothelial cells” are flat cells that line the lumen of blood vessels, and have various functions such as regulation of vascular tone and vascular permeability, angiogenesis, anti-inflammatory, and blood coagulation promotion. Large blood vessels at the level of arteries and veins have a three-layered structure consisting of the intima, the media, and the adventitia, each of which is mainly composed of vascular endothelial cells, smooth muscle cells, and fibroblasts. On the other hand, in small blood vessels at the capillary level, the luminal structure of vascular endothelial cells is surrounded by pericytes. In mature capillaries, pericytes share the basement membrane with vascular endothelial cells and are embedded therein. In recent years, mutual cell signaling between pericytes and vascular endothelial cells regulates differentiation and proliferation, and is important for the maturation, stabilization, and maintenance of capillaries, formation of basement membrane, and deposition of extracellular matrix. known to play a role.

Vascular endothelial cells that can be combined with the pericytes of the present invention or the pharmaceutical composition of the present invention are not particularly limited, but in one embodiment, primary vascular endothelial cells or induced differentiation from pluripotent stem cells or the like vascular endothelial cells. Vascular endothelial cells used in combination with the pericytes of the present invention or the pharmaceutical composition of the present invention are not particularly limited, but in one embodiment, primary vascular endothelial cells, or blood vessels differentiated from pluripotent stem cells or the like endothelial cells. In the angiogenesis therapy of the present invention, when the pericytes of the present invention and vascular endothelial cells are used in combination, the vascular endothelial cells are administered simultaneously with the administration of the pericytes of the present invention, or before or after the administration of the pericytes of the present invention. can be administered.

"Primary vascular endothelial cells" means vascular endothelial cells directly collected from an individual organism, or primary cultured cells or passaged cells obtained by culturing and proliferating the vascular endothelial cells in vitro. Although the primary vascular endothelial cells are not particularly limited, in certain embodiments, they are human primary vascular endothelial cells. Human primary vascular endothelial cells include, for example, primary human vascular endothelial cells that have the same or substantially the same human leukocyte antigen (HLA) genotype as that of a human patient, or a recipient individual from the viewpoint that rejection does not occur. It is desirable to use vascular endothelial cells. Here, the term “substantially identical” means that the HLA genotypes match the transplanted vascular endothelial cells to the extent that the immunosuppressive agent can suppress the immune reaction. A vascular endothelial cell having an HLA type corresponding to 3 loci of -B and HLA-DR or 4 loci including HLA-C. Vascular endothelial cells that can be used or combined with the pericytes of the present invention or the pharmaceutical compositions of the present invention are, in one embodiment, primary human vascular endothelial cells.

The "vascular endothelial cells differentiated from pluripotent stem cells or the like" that can be combined with the pericytes of the present invention or the pharmaceutical composition of the present invention are not particularly limited, but for example, Ikuno T et al., Pros One, (2019), 12: e0173271 and Cho SW et al., Circulation, (2007), 116: 2409-2419, vascular endothelial cells produced by the method described can be used. Vascular endothelial cells that can be used or combined with the pericytes of the present invention or the pharmaceutical composition of the present invention are, in one embodiment, vascular endothelial cells induced to differentiate from human pluripotent stem cells or the like.

Specific examples are provided herein for reference to gain a further understanding of the invention, but these are for illustrative purposes and do not limit the invention.

Example 1: Establishment of primary human pericyte derived from skeletal muscle A portion of the human quadriceps muscle was soaked in PBS, and the muscle was finely cut along the fiber using a scalpel and tweezers. The cut muscle fibers were placed in a 50 mL centrifuge tube (Corning, 352070), 15 mL of collagenase solution (see below) was added, allowed to stand for 3 minutes, and the supernatant was removed. The above operation was repeated two more times. Next, the muscle fiber subjected to the above operation was placed in another 50 mL centrifuge tube, 15 mL of collagenase solution was added, and the tube was allowed to stand in a warm bath at 37° C. for 1 hour. A cell suspension other than the muscle fibers was collected and designated as cell suspension 1. The collagenase solution was again added in two portions of 15 mL each, and each addition of the collagenase solution was collected in the same manner as the cell suspension 1 to obtain a cell suspension 2 and a cell suspension 3. Cell suspensions 1, 2 and 3 were each passed through a 100 μm cell strainer (Corning, 352360), and the resulting cell suspensions were centrifuged at 300 g at 4° C. for 5 minutes. Remove the supernatant, add 20 mL of pericyte establishment medium (see below) to each centrifuge tube, divide into 2 collagen-coated dishes (Corning, 356450), and incubate at 37°C, 5% CO ₂ , 5% O ₂ . Cultured under atmosphere. Cells from cell suspensions 1, 2 and 3 are referred to below as condition 1, 2 and 3 cells respectively. On the 3rd, 6th and 8th days of culture, each culture supernatant was removed and 10 mL of pericyte establishment medium was added. For the cells in Condition 1, remove the culture supernatant on the dish on day 13 of culture, wash with 5 mL of PBS, add 1 mL of cell dissociation reagent (TrypLE Express, ThermoFisher Scientific, 12604013), and place on a 37°C plate for 5 minutes. Let stand for 1 minute. 4 mL of pericyte establishment medium was added to the dish to collect the cell suspension, which was then centrifuged at 300 g at room temperature for 5 minutes. After removing the supernatant, 5 mL of cell banker 1 (Takara Bio, CB011) was added, and 1 mL portions were dispensed into cryotubes and stored in liquid nitrogen.

On the 10th day of culture, the culture supernatants of the dishes of the cells of conditions 2 and 3 were removed, and after washing each dish with 5 mL of PBS, 1 mL of TrypLE Express was added to each dish, and allowed to stand on a 37° C. plate for 5 minutes. Add 4 mL of pericyte establishment medium to each dish to collect the cell suspension, mix the cell suspensions of conditions 2 and 3 together in the same centrifuge tube, and centrifuge at 300 g at room temperature for 5 minutes. separated. After removing the supernatant, 5 mL of cell banker 1 was added, and 1 mL portions were dispensed into cryotubes and stored in liquid nitrogen. At a later date, add 9 mL of pericyte growth medium (see below) to a 50 mL centrifuge tube, add 1 mL of cell banker 1 solution containing the thawed cells of conditions 2 and 3 above, and incubate at 300 g at 4°C for 5 minutes. Centrifuged. The supernatant was removed, 20 mL of pericyte growth medium was added to the centrifuge tube, and the cell suspension was recovered. The collected cell suspension was divided into 2 collagen-coated dishes and cultured at 37° C., 5% CO ₂ , 5% O ₂ atmosphere. On day 3 of culture, the culture supernatant of each dish was removed, and 10 mL of pericyte growth medium was added. On the next day, the culture supernatant was removed, and after washing with 5 mL of PBS, 2 mL of a cell dissociation reagent (Accutase (registered trademark), Innovative Cell Technologies, AT104) was added, and allowed to stand on a 37°C plate for 5 minutes. 8 mL of pericyte growth medium was added to each dish, and the cell suspension was collected in a centrifuge tube and centrifuged at 300 g at room temperature for 5 minutes. The supernatant was removed, 100 µL of PBS was added to the centrifuge tube, 20 µL of a blocking reagent (FcR Blocking Reagent, Miltenyi Biotec, 130-059-901) was added, and the tube was allowed to stand on ice for 15 minutes. Subsequently, 20 μL each of anti-human ALP (Alkaline Phosphatase) antibody (R&D Systems, FAB1448P) and anti-CD56 APC antibody (Miltenyi Biotec, 130-090-843) were added and allowed to stand on ice for 20 minutes. After washing twice with PBS, the cells were resuspended in PBS, and ALP(+) and CD56(-) cells (that is, primary human pericytes) were sorted using a cell sorter (SH800S, Sony). The collected cells were suspended in a pericyte growth medium, seeded on a collagen-coated dish, and cultured.

[Collagenase solution]
It was prepared by dissolving 100 mg of Collagenase, Type II (ThermoFisher Scientific, 17101015) in 200 mL of TrypLE select (ThermoFisher Scientific, 12563029).

[Pericyte establishment medium]
The composition is as follows.
・92% MegaCell Dulbecco's Modified Eagle's Medium (Sigma-Aldrich, M3942)
・5% FBS (Sigma-Aldrich)
・1% GlutaMax (ThermoFisher Scientific, 35050061)
・1% MEM Non-essential Amino Acid Solution (Sigma-Aldrich, M7145)
・1% Penicillin-Streptomycin (Sigma-Aldrich, P0781)
・100 μM 2-Mercaptethanol (ThermoFisher Scientific, 21985023)
・5ng/mL FGF-basic (154a.a.), Human, Recombinant (Peprotech, 100-18B)

[Pericyte growth medium]
The composition is as follows.
・77% MegaCell Dulbecco's Modified Eagle's Medium
・20% FBS
・1% GlutaMax
・1% MEM Non-essential Amino Acid Solution
・1% Penicillin-Streptomycin
・100 μM 2-mercaptethanol
・5ng/mL Animal-free Recombinant Human FGFbasic-TS (Proteintech, HZ-1285)

Example 2: Preparation of lentiviral vector for bFGF gene transfer A cell line for lentiviral packaging (Lenti-X 293T Cell Line, Takara Bio, 632180) was grown in 10 mL of 293T growth medium (see below) per 10 cm dish. 5×10 ⁶ cells were seeded, and the cells seeded in 4 dishes were cultured at 37° C. in a 5% CO ₂ atmosphere. On the next day, add 28 μL of Lentiviral High Titer Packaging Mix (Takara Bio, 6194) and 15 μL of bFGF insertion plasmid (see below) to 6 mL of D-MEM (Fujifilm Wako Pure Chemical Industries, Ltd., 045-30285), mix, and leave at room temperature for 5 minutes. placed. Further, 180 μL of a transfection reagent (TransIT-293 Transfection Reagent, Takara Bio Inc., MIR2704) was added thereto, mixed, and allowed to stand at room temperature for 30 minutes (hereinafter referred to as “transfection solution”). 1.5 mL of Transfection solution was added to each of the 4 dishes per 10 cm dish seeded with the Lenti-X 293T Cell Line the day before. The next day, the supernatant of each dish was removed and 10 mL of 293T growth medium was added. Two days later, the culture supernatant was collected and centrifuged at room temperature at 300 g for 5 minutes to remove detached cells. The supernatant was recovered, 1/3 of the supernatant was added with a lentivirus concentration reagent (Lenti-X Concentrator, Takara Bio, 631231), and centrifuged at 500 g at 4°C for 45 minutes. After removing the supernatant, 200 μL of D-MEM was added to obtain a lentiviral suspension for bFGF gene transfer.

[293T growth medium]
The composition is as follows.
・D-MEM
・10% FBS
・1% GlutaMAX

[bFGF insertion plasmid]
Plasmid (pLe6 -Bmp-bFGF: SEQ ID NO: 5) was produced.

Example 3: Preparation of bFGF gene-introduced human primary pericytes The human primary pericytes established in Example 1 were grown in a pericyte growth medium at 1 × 10 ⁵ cells/well in a collagen-coated 6-well plate (Iwaki, 4810). -010) were seeded in the top two wells. Two days later, 1.2 μL of 10 mg/mL polybrene solution (Nacalai Tesque, 12996-81) and 62 μL of lentiviral suspension for bFGF gene transfer prepared in Example 2 were added to 3 mL of pericyte growth medium, mixed, and incubated at room temperature. Let sit for 5 minutes (suspension A). After removing the supernatant of cultured primary human pericytes, 1.5 mL of Suspension A was aspirated per well and added to each of the two wells. The plate was centrifuged at room temperature at 1200 g for 60 minutes and then cultured at 37° C., 5% CO ₂ , 5% O ₂ atmosphere. On the next day, the supernatant was removed, and after washing with PBS nine times, 1 mL of Accutase was added and allowed to stand on a 37°C plate for 5 minutes. 4 mL of pericyte growth medium was added to the dish to collect the cell suspension, and the cell suspension for 2 wells was collected in the same centrifuge tube and centrifuged at 300 g at room temperature for 5 minutes. The supernatant was removed, 10 mL of pericyte growth medium was added, and the collected cells were seeded on a collagen-coated dish and cultured. Cultivation was performed at 37° C., 5% CO ₂ , 5% O ₂ atmosphere. On the following day, a 10 mg/mL Blasticidin S solution (Fuji Film Wako Pure Chemical Industries, Ltd., 026-18711) was added to the collagen dish to a final concentration of 2.5 μg/mL, and the culture was continued. Cells proliferated through this procedure were used as bFGF gene-introduced primary human pericytes.

Example 4: Quantitation of bFGF Expression Level in bFGF Gene-Introduced Human Primary Pericytes Using the culture medium, the cells were seeded on a collagen-coated dish at 3×10 ⁵ cells/dish and cultured at 37° C. in an atmosphere of 5% CO ₂ and 5% O ₂ . On day 3 of culture, the culture supernatant was collected and passed through a syringe filter (IWAKI, 2053-025). The concentration of bFGF in the supernatant passed through the syringe filter was measured using an ELISA kit (Human FGF basic Quantikine ELISA KIT, R&D Systems, DFB50). The bFGF concentration was measured according to the protocol attached to the kit.

In the control human primary pericytes, bFGF expression could not be detected, whereas in the bFGF gene-introduced human primary pericytes, very high bFGF expression was observed (Fig. 1).

Example 5: Qualitative evaluation of angiogenic potential of bFGF gene-introduced human primary pericytes ) and cultured at 37°C in a 5% CO ₂ atmosphere. In addition, the human primary pericytes (control) established in Example 1 and the bFGF gene-introduced primary human pericytes prepared in Example 3 were each cultured on a collagen-coated dish using a pericyte growth medium. After confirming that each of the three types of cells proliferated to a confluent state, the supernatant was removed, and after washing with PBS, 2 mL of Accutase was added and allowed to stand on a 37° C. plate for 5 minutes. To each dish was then added 8 mL of endothelial cell growth medium for HUVECs and pericyte growth medium for human primary pericytes and bFGF-transduced human primary pericytes. Cell suspensions were collected from each dish and centrifuged at 300 g at room temperature for 5 minutes to remove the supernatant. suspended in growth medium. 5.5×10 ⁵ HUVECs were added to each of 9 1.5 mL tubes (Eppendorf, 0030120.086), 3 of which were human primary pericytes and 3 of them were bFGF transgenic human primary pericytes. were added and mixed at 5.5×10 ⁵ per tube. Each tube was centrifuged at 300 g at 4°C for 5 minutes, the supernatant was removed, and after washing once with PBS, the tube was centrifuged again under the same conditions and the supernatant was removed. Next, 400 μL of extracellular matrix (Matrigel (registered trademark) Growth factor reduced, Corning, 356231, hereinafter referred to as “Matrigel”) was added to each tube, mixed on ice, and the Matrigel containing the cells was attached with a 25 gauge needle. Aspirated with a syringe. To 79 mL of physiological saline (Otsuka Pharmaceutical Factory), 3 mL of Domitol (Nippon Zenyaku Kogyo), 8 mL of Dormicum Injection (Maruishi Pharmaceutical), and 10 mL of Betrufal (Meiji Seika Pharma) were added to prepare a mixed anesthetic solution. Nine NOG mice (NOD.Cg-Prkdc ^scid Il2rg ^tm1Sug /ShiJic mice, In-Vivo Science) were each intraperitoneally administered with 300 μL of the three kinds of mixed anesthetic solution. Prepared HUVEC alone (HUVEC), HUVEC and control human primary pericyte (HUVEC), HUVEC and bFGF-transduced human primary pericyte (bFGF-Primary pericyte/HUVEC) were administered to each of three anesthetized mice. A full dose of Matrigel containing After 14 days, the administered Matrigel was collected from each mouse and photographed (Fig. 2).

Matrigel containing bFGF-transfected human primary pericytes (bFGF-Primary pericyte/HUVEC in Fig. 2) showed more angiogenesis than control Matrigel containing human primary pericytes (Primary pericyte/HUVEC in Fig. 2). . In addition, no angiogenesis was observed in Matrigel containing only HUVEC (HUVEC in FIG. 2).

Example 6: Quantitative evaluation of angiogenesis exhibited by bFGF-transfected human primary pericytes Each Matrigel collected by the procedure of Example 5 was placed in a 2 mL tube (Eppendorf, 0030120.094) and cut several times with dissecting scissors. One stainless steel bead (Qiagen, 69989) was added thereto, and 350 μL of 0.1% Brij (registered trademark) L23 solution (Sigma-Aldrich, B4184) was added. Matrigel was crushed using TissueLyser II (Qiagen, 85300), centrifuged at 10,000 g at 4°C for 5 minutes, and the supernatant was recovered. The hemoglobin concentration in the recovered supernatant was measured using QuantiChrom Hemoglobin Assay Kit (BioAssay Systems, DIHB-250). The method followed the product protocol. As a statistical analysis test, a t-test was performed between two groups of HUVEC and control human primary pericyte (Primary pericyte/HUVEC), and between HUVEC and bFGF gene-introduced human primary pericyte (bFGF-Primary pericyte/HUVEC).

The hemoglobin concentration in Matrigel containing bFGF gene-introduced human primary pericytes was significantly higher than that in Matrigel containing control primary pericytes (Fig. 3).

Example 7 Evaluation of Improvement in Blood Flow of bFGF Gene-Transduced Human Primary Pericytes in Lower Limb Ischemia Model 300 μL of a three-kind mixed anesthetic solution having the same composition as that used in Example 5 was administered intraperitoneally to NOG mice. After anesthesia, body hair around the left lower leg was removed using depilatory cream. Thereafter, 300 μL of anti-sedan preparation solution prepared by adding 150 μL of anti-sedan (Nippon Zenyaku Kogyo) to 24.8 mL of physiological saline was subcutaneously administered to awaken the NOG mice. The next day, the NOG mouse was again anesthetized with the three kinds of mixed anesthetic solution in the same manner as the previous day, placed on its back under a stereoscopic microscope, and the skin of the left lower limb was cut open to expose the femoral artery and vein and the saphenous artery and vein. rice field. After ligating the blood vessels branching from the femoral artery and vein, the femoral artery and vein and the saphenous artery and vein were excised and the skin was sutured. Subsequently, the NOG mouse was placed prone, the skin near the gastrocnemius muscle was incised, and the marginal vein was cut. The skin was sutured again, and 300 μL of the anti-sedan preparation solution was administered subcutaneously to awaken the NOG mice. Two weeks after the operation, the NOG mice were anesthetized with the above three kinds of mixed anesthetic, kept warm for 10 minutes on a warming plate at 36°C, and the blood flow in the lower extremities was measured with a blood flow imaging device (moorLDI2-IR, moor instruments). and analyzed. The blood flow signal value of the operated ischemic limb was divided by the blood flow signal value of the non-operated normal limb to calculate the blood flow ratio (% conversion). At the same time, the number of necrotic nails on the toes of the NOG mice whose blood flow was measured was recorded. Among the multiple lower limb ischemia model mice that underwent the above procedure, mice with a blood flow ratio of 20% to 40% and with 4 or 5 necrotic nails were selected as evaluation mice. , Grouping was performed so that the average blood flow ratio was the same between the two groups. The next day, the NOG mice divided into two groups were anesthetized with a three-kind mixed anesthetic, and the skin of the left lower leg was cut open to expose the muscle. In one group, 3 × 10 ⁶ bFGF-transduced primary human pericytes suspended in 100 μL of Megacell Dulbecco's Modified Eagle's Medium were placed on the sole of the left lower leg at a total of 9 points from the left lower femoral muscle to the gastrocnemius muscle. 10 µL each was administered to a total of 10 sites (cell-administered group; 8 animals). As a control, only Megacell Dulbecco's Modified Eagle's Medium was similarly administered to another group (medium-administered group; 7 animals). 2, 4, and 6 weeks after administration of the cells or medium, the blood flow in the lower extremities was analyzed using the blood flow imaging device moorLDI2-IR as described above, and the signal value of the blood flow in the ischemic limb was evaluated as normal. The blood flow ratio (% conversion) (Fig. 4, left) and AUC (Area Under Curve) up to 6 weeks after cell administration were calculated by dividing by the limb blood flow signal value (Fig. 4, right). Here, AUC is the area under the polygonal line drawn when time is plotted on the X-axis and blood flow ratio is plotted on the Y-axis. A t-test was performed as a statistical analysis test.

In lower extremity ischemia model mice, administration of bFGF gene-introduced primary human pericytes significantly improved blood flow.

Example 8: Differentiation induction from ES cells to early mesodermal cells Add 230 μL of Matrigel human ES cell optimized matrix (Corning, 354277) to 25 mL of DMEM/Ham F12 medium (Nacalai Tesque, 11581-15) and place in a 6-well plate. (Iwaki, 3810-006) is added to 3 wells of 1.5 mL each and allowed to stand at room temperature for 3 hours to prepare a Matrigel-coated plate. Human ES cells were seeded on the Matrigel-coated plate and cultured using STEMdiff Mesoderm induction medium (STEMCELL Technologies, 05220) at 37°C in a 5% CO ₂ atmosphere to induce differentiation from ES cells to early mesoderm cells. I do. Flow cytometry is used to confirm whether the obtained cells are differentiated into early mesoderm cells. Specifically, CD140α and APLNR were selected as cell surface markers of early mesodermal cells, and the ratio of CD140α(+) and APLNR(+) cells was increased by flow cytometry using antibodies against each cell surface marker. By confirming this, it is confirmed that differentiation induction from ES cells to early mesoderm has progressed.

Example 9: Spheroid formation from early mesoderm cells 1.7 × 10 ⁵ early mesoderm cells obtained in Example 8 were documented (Vodyanik MA et al., Cell Stem Cell, (2010), 7: 718-729 ), and cultured on an EZSPHERE (registered trademark) dish (Iwaki, 11-0434) at 37°C in an atmosphere of 5% CO ₂ and 5% O ₂ . When spheroids are formed, pass the spheroid-containing culture medium through a 100 μm cell strainer to collect spheroids with a size of 100 μm or larger.

Example 10 Differentiation Induction from Spheroids to Pericyte-Like Cells Based on US Pat. Specifically, all the spheroids collected in Example 9 are suspended in a pericyte differentiation-inducing medium (see below), seeded on a dish coated with Fibronectin and Human type 1 Collagen, and cultured. When monolayer cell proliferation is observed on the bottom of the dish, the supernatant is removed, washed with PBS, then Accutase is added, and the cells are detached. The cell suspension is harvested in the dish using pericyte growth medium (see Example 1) and centrifuged. The supernatant is removed, a pericyte growth medium is added, seeded on a collagen-coated dish, and further cultured.

[Pericyte differentiation induction medium]
The composition is as follows:
- 50% Stemline® II Hematopoietic Stem Cell Expansion Medium (Sigma-Aldrich, S0192)
・50% Human Endothelial SFM (ThermoFisher Scientific, 11111044)
・1% GlutaMax
・0.05% Ex-CYTE NZ Growth Enhancement Media Supplement (Merck, 81150N)
・100μM Monothioglycerol (Fujifilm Wako Pure Chemical Industries, 195-15791)
・10ng/mL Animal-free Recombinant Human FGFbasic-TS
・50ng/mL Recombinant Human PDGF-BB Protein (R&D Systems, 220-BB)

Example 11: Introduction of bFGF Gene into Human ES Cell-Derived Pericyte-Like Cells For the human ES cell-derived pericyte-like cells prepared in Example 10, the lentivirus for bFGF gene transfer was prepared in the same manner as in Example 2. Using the suspension, the bFGF gene is introduced into human ES cell-derived pericyte-like cells in the same manner as in Example 3 for human primary pericytes.

Example 12: Quantitation of bFGF Expression Level in bFGF Gene-Transduced Human ES Cell-Derived Pericyt-Like Cells The bFGF expression level of the human ES cell-derived pericyte-like cells is quantified in the same manner as in Example 4. The bFGF gene-introduced human ES cell-derived pericyte-like cells exhibit a higher bFGF expression level than the control.

Example 13: Evaluation of angiogenic potential of bFGF transfected human ES cell-derived pericyt-like cells Evaluation of the angiogenic potential of bFGF transfected human ES cell-derived pericyt-like cells It can be carried out in the same manner as in Examples 5 and 6 in which the performance was evaluated. The bFGF-transfected human ES cell-derived pericyte-like cells exhibit higher angiogenic potential than the control human ES cell-derived pericyt-like cells.

Example 14: Evaluation of blood flow improvement of bFGF gene-transduced human ES cell-derived pericyte-like cells in lower limb ischemia model It can be performed in the same manner as in Example 7, in which blood flow improvement in bFGF gene-introduced human primary pericytes was evaluated using ischemia model mice. Administration of bFGF gene-introduced human ES cell-derived pericyte-like cells exhibits a high blood flow improvement effect in the model mice.

The pericytes into which the bFGF gene of the present invention has been introduced have excellent angiogenic activity and can be used for angiogenic therapy for severe lower extremity ischemia.

The nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing is the gene sequence of human bFGF shown in GenBank Accession Number: M27968.1, and the amino acid sequence shown in SEQ ID NO: 2 is shown in GenBank Accession Number: AAA52448.1. is the amino acid sequence of human bFGF. In addition, the description of "Artificial Sequence" is provided under the number heading <223> in the sequence listing below. Specifically, the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing is a nucleotide sequence that encodes the amino acid sequence of human bFGF analog shown in SEQ ID NO: 4. The nucleotide sequence shown in SEQ ID NO: 5 in the sequence listing is the nucleotide sequence of the human bFGF expression plasmid (pLe6-Bmp-bFGF) used in the Examples of the present application.

Claims

A pericyte into which the basic fibroblast growth factor (bFGF) gene has been introduced.
The pericyte according to claim 1, wherein the pericyte is primary pericyte.
The pericytes according to claim 1, wherein the pericytes are pericyte-like cells differentiated from pluripotent stem cells.
The pericyte according to claim 3, wherein the pluripotent stem cells are human pluripotent stem cells.
The pericytes according to claim 3 or 4, wherein the pluripotent stem cells are embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells).
A pharmaceutical composition for angiogenesis therapy, comprising the pericyte according to any one of claims 1 to 5.
The pharmaceutical composition according to claim 6, wherein the angiogenesis therapy is treatment of critical limb ischemia.
The pharmaceutical composition according to claim 6 or 7, which is used in combination with vascular endothelial cells.
A pharmaceutical composition for angiogenesis therapy, comprising a combination of the pericytes and vascular endothelial cells according to any one of claims 1 to 5.
The pharmaceutical composition according to claim 9, wherein the angiogenesis therapy is treatment of critical limb ischemia.
A method for producing pericyte according to any one of claims 1 to 5.
An angiogenesis therapy comprising administering a therapeutically effective amount of the pericyte according to any one of claims 1 to 5 to a subject.
The angiogenesis therapy according to claim 12, further comprising administering vascular endothelial cells.
The angiogenesis therapy according to claim 12 or 13, which is a treatment for critical limb ischemia.
Use of the pericyte according to any one of claims 1 to 5 in the manufacture of a pharmaceutical composition for angiogenesis therapy.
Use of the pericyte according to any one of claims 1 to 5 in the manufacture of a pharmaceutical composition for treating critical limb ischemia.
The pericyte according to any one of claims 1 to 5, for use in angiogenesis therapy.
Pericytes according to any one of claims 1 to 5, for use in treating critical limb ischemia.